Project Summary The Food and Drug Administration (FDA) regulates pet food, requiring it is safe to eat, contains no harmful substances, is produced using sanitary conditions, and is truthfully labeled. However, the foods do not need pre-market approval because they are made with ingredients considered safe. Pet food adulteration has been shown to occur in plants during processing, resulting in illness in animals and humans related to foodborne pathogens. Plant processing may also result in cross-contamination between products used for pet food production. This can result in pet foods having unintended ingredients, leading to false labelling. Whether intentional or not, adulteration of pet food with DNA from unlabeled genera has been previously detected. Current methods used to detect foodborne pathogens are labor-intensive and may take several days to obtain actionable results. Additionally, samples likely have to been sent to several different labs for comprehensive testing (e.g. for virus, bacteria, and parasites) and for detection of the genus of the meat source in the food. Additional testing capabilities, including comprehensive methods for rapid detection and initial characterization, would be beneficial for rapid recall of adulterated food. We propose to develop a targeted next-generation sequencing (NGS) panel, which incorporates primers for multiple pathogens associated with foodborne illness, for detection and initial characterization of the involved pathogen(s), and for characterization of the meat included in the product. Primers can be incorporated into the NGS workflow for multiplex PCR prior to sequencing. Use of primers allows for standardization of the NGS protocol for consistent performance across multiple labs and eases the use and implementation of the technology by those already familiar with PCR and Sanger sequencing. This renders implementation of NGS relatively simple in the diagnostic lab setting, even though NGS bioinformatics pipelines still need further development and validation for standardization across labs. The simplistic analysis of the data generated from a targeted NGS panel mitigates this limitation. Additionally, as a result of the initial PCR amplification steps, the test can be performed directly from the clinical sample or following an enrichment process. All of these advantages suggest that targeted NGS can be used to provide a comprehensive diagnostic assay for the detection of foodborne pathogens with initial pathogen characterization and detection of meat source genera, that has a more rapid turn-around time than currently used methods.